The long-term goal of the grant over the past 15 years has been to understand both the Cl- channel and regulatory functions of CFTR. In the previous budget period, we focused specifically on the role of the PDZ binding domain and asked whether CFTR is a member of a macromolecular complex. We discovered a new protein, CAL that tethers CFTR within the Golgi and targets CFTR for degradation in the lysosome. We also studied NHE-RF and CAP70 how these PDZ domain-containing proteins function in relation to CAL, thus addressing the issue of why multiple PDZ domain-containing proteins bind to CFTR. The present proposal posits that the role of the interaction of CFTR with the PDZ domain of CAL is to regulate the amount of CFTR at the plasma either by tethering it at the Golgi, and targeting CFTR for degradation or allowing it to process to the plasma membrane. The proposal hypothesizes further, that the CAL-mediated decision whether CFTR is destined to move to the plasma membrane or not is determined by two associated proteins that bind to CAL, TC10 and syntaxin 6. The grant suggests further that the CAL-CFTR interaction with these two associated proteins has an important role in how CFTR responds to bacterial toxins that lead to diarrheal diseases and infection in the airways. The overall goal is to address the following three questions: Is the trafficking of mature CFTR to the plasma membrane regulated by TC10? Is the trafficking of mature CFTR to the plasma membrane regulated by Syntaxin6? How do bacterial toxins function in the trafficking of CFTR to the plasma membrane? Relevance: The proposal represents a new area of investigation with implications both for our understanding of diarrheal diseases involving toxins that affect Rho-GTPases and for Pseudomonas infection in lung where the toxin ExoS, may alter CFTR trafficking to the membrane. Once the critical pathways are identified and their roles established, it may be possible to develop drugs to therapeutically alter the switching mechanisms that determine CFTR's fate.